1. Field of the Invention
This invention relates to methods and apparatus for preparing biological suspensions for long term preservation in the dry state for later use. These methods avoid deleterious modifications of the biological ultrastructure during the preservation process. More particularly, the present invention relates to the preservation of microscopic biological materials which are found or placed in a suspension prior to treatment.
In its preferred form, the method of this invention includes the steps of preparing a cryosolution by treating a biological material with a protectant or combination of protectants, cooling, storing under temperature conditions which preclude devitrification or recrystallization, and drying under controlled temperature, vacuum and condenser conditions. The biological material can be rehydrated prior to use if desired.
2. Description of the Related Art
The desire to preserve biological materials at ambient conditions for future use has existed for a long time in the scientific community. For example, the dry preservation of erythrocytes has been attempted by several groups over the last 30 to 40 years. The ability to dry the erythrocytes and reconstitute them at a later date at ambient conditions would have far reaching benefits during times of disaster, wars, and in remote locations. It would be a significant advancement in the art to be able to store dried, powdered erythrocytes in a sterile container such that they could be reconstituted by simply adding an appropriate buffered solution prior to use.
Similarly, freeze drying of cultured mammalian cells has been attempted without success. With the advent of biotechnology and the identification of functionally important cells, e.g., hepatocytes and pancreatic islets, a method of prolonged storage under ambient conditions would be invaluable.
Many complex macromolecules, either antibodies or recombinant proteins which are to be used therapeutically, show reduced activity following freezing or freeze drying. The development of methods to enable prolonged ambient storage without significant loss of activity is an important component in their ultimate therapeutic effectiveness.
Further, the necessity to control or abate viral infections has been a major medical concern for decades. Basically, the eradication and/or control of viral infections involves the production of a variety of different and highly potent vaccines.
Quantitative and qualitative production of pure and potent viral antigens or immunogens is difficult as well as expensive. This is usually due to the fact that many viruses replicate very slowly, if at all, in controlled cell culture environments. If the virus of choice seldom replicates in the test tube, then the production of the specific viral particle becomes less favorable and effective, as well as more expensive. Therefore, it is essential that, once vaccine material is produced, its biological activity be preserved for an extended period of time.
One feature common to all these examples is that the initial configuration of the sample is a microscopic biological material in the form of a suspension or emulsion. Once an effective suspension has been produced, preserving and storing this material is of utmost importance.
The field of cryopreservation and dry stabilization is rapidly developing and advancing. Incorporated herein by reference is U.S. Pat. No. 4,865,871, issued Sep. 12, 1989 which describes an apparatus and method for the cryopreservation of a biological tissue sample. The method comprises treating a biological tissue sample with a cryoprotectant which raises the glass transition temperature range of the sample, vitrifying the tissue sample under cryogenic temperature conditions at a specific rate to preclude the formation of significant resolvable ice crystals, equilibrating the depressurized, vitrified tissue in a sample holder and then dehydrating the tissue sample by molecular distillation by the addition of energy until substantially no vaporized water can be detected in the atmosphere surrounding the tissue sample.
The first critical processing step in cryopreservation must be performed in such a way as to satisfy two essential criteria. First, the biological material must not undergo irreversible damage due to the multiplicity of changes which occur within a sample during cooling. These changes include mechanical damage due to ice formation, cell-to-cell fusion due to the decrease in the solute volume available, and changes in acidity (pH) and salt or solute concentrations due to the segregation of solute and water. Second, the condition of the sample following cooling must be compatible with subsequent drying and reconstitution. This includes such parameters as sample size, ice forms created, and the nature and final concentration of additives or excipients.
In satisfying these two criteria, the cryopreservation process represents a balance between the use of cryoprotective agents (CPA's) to minimize changes during freezing by chemically increasing the volume of the ice free zone for a given cooling rate, and the cooling rate itself. Several potential combinations are possible, some of which are summarized in the following table:
Approximate Final CPA Cooling Mode Cooling Rate Concentration Ultra Rapid 300,000.degree. C./sec. Zero Rapid 50,000-100,000.degree. C./sec. Low Intermediate 500-2,500.degree. C./sec. Intermediate Slow 1-20.degree. C./min. High
Inherent in most processes for cryopreparation and freezing of biological materials is the concomitant artifact creation, sample shrinkage and resultant damage to and modification of sample characteristics. These sample characteristic modifications, whether in the form of artifacts or the like, must be controlled and minimized. The acceptable limits for sample modification are defined by the anticipated end use of the cryoprepared material. For example, slow rate cooling results in extremely high final concentrations of CPA to which the sample is exposed. This results in potential deleterious effects at each step of the process. These include toxicity during cooling, incompatibility during drying and severe osmotic stresses during rehydration.
One of the problems which has been encountered in cryopreservation has been the lack of an effective and efficient apparatus and method for rapidly cooling suspensions of biological materials. Some prior art processes have utilized air guns or air brushes to generate microdroplets which are then cooled. However, these devices cause shear stresses which damage the cellular material. Additionally, in the prior art processes, the microdroplets were sprayed directly into a liquid cryogen. This posed problems in collection of the frozen microdroplets and also had the potential for contaminating the sample. Accordingly, there has existed in the art a need for a method and apparatus for the cryopreparation of a suspension of biological materials which was practical, cost effective and sterile.
Various processes have been utilized in the past for drying frozen biological samples. These include freeze drying and molecular distillation drying. Examples of apparatus and methods used in molecular distillation drying are disclosed in U.S. Pat. Nos. 4,510,169, 4,567,847, 4,676,070, 4,799,361, 4,865,871 and 4,964,280.
Thus, it would be an important advancement in the art to provide methods and apparatus of cryopreparing and dry stabilizing biological samples without overt disruption or destruction of the morphological characteristics of the ultrastructure of the sample. Such methods and apparatus should provide for the cryostabilization of microscopic, biological samples by dehydrating the samples, in which the water molecules are maintained in a predetermined optimal ice phase, without creating unnecessary artifacts and resultant undesired ultrastructural and morphological damage while the water is being removed.